A wide variety of important procedures in biological research and biotechnology involve getting certain materials in to and out from cells. These procedures include gene cloning, DNA library construction, cell fusion, production of cloned proteins, tests on the effects of inserting certain materials into cells, and collection of certain materials found inside cells. Furthermore, these procedures must often be carried out on a large number of independent samples which must not be cross-contaminated. Electroporation, the transient formation of small holes in dielectrics in response to electric fields, provides a means of perforating the cell membrane for accomplishing these procedures (Neumann et al., 1982, EMBO J. 1:841-845).
Prior art electroporation equipment is comprised of large high voltage power supplies capable of producing substantial current at up to 2500 volts, such as that disclosed by Ragsdale on Jun. 7, 1988 in U.S. Pat. No. 4,750,100. Power supplies generating various waveforms, including rectangular, unipolar, bipolar, exponential decay, and radio frequency, are attached via a switching and/or timing means to a sample chamber in which electrodes are spaced to give a field strength appropriate to form pores in the cells of interest (Tekle et al., 1991, PNAS. 88:4230-4234). Existing devices such as that disclosed in U.S. Pat. No. 4,800,163 by Hibi et al. in 1989, are large, expensive, complex, and are more efficient for bulk industrial processing than for the processing of the dozens of individual samples more common in research laboratory work. In working with such samples, which must not be cross-contaminated, it is necessary to replace the electrode assembly with a clean, sterile electrode assembly before each sample is processed. It is an object of the present invention to provide an economical, easily and quickly replaceable, electrode assembly and associated apparatus especially well suited for research laboratory use. Because of the potentially lethal voltage/current combination, sample chambers are usually hidden in protective enclosures with safety interlocks to keep the user at a distance from the high voltage. While such an apparatus provides a reliable means of delivering voltage to a sample, it occupies considerable space and requires complex and time-consuming manipulations to insert and remove samples. Aside from the tedium and high labor cost, there is another significant problem with the prior art apparatus. As described by Dower in U.S. Pat. No. 4,910,140, Electroporation of Prokaryotic Cells, on Mar. 20, 1990, the efficiency of recovering live cells containing molecules of interest falls rapidly as time increases.
The improvements to electroporation of the present invention eliminate the tedium of a human operator as well as increase the survival of electroporated samples due to reduced time under hostile conditions. Some prior art instruments provide numerical feedback relating to current, voltage and wave shape parameters of the delivered energy. The present invention simplifies operation by providing go/no go indicator lights instead of confusing numerical information.
Because only brief pulses of high current are required, a number of options other than the typical bulky continuous duty high voltage power supply are disclosed. In the preferred embodiment of the present invention, a small Direct Current inverter type transformer is used in "flyback" mode to store charge in a capacitor for subsequent discharge. In electrotransfection experiments with E. coli bacteria, efficient transformation was observed using Alternating Current sources including 120 volt 60 hertz line power. Therefore an alternate embodiment of the present invention eliminates the high voltage supply altogether and substitutes the timed application of A.C. line current directly to the sample. The rapid mechanical deformation of piezo-electric crystals provides a high voltage pulse. Therefore in a third embodiment of the present invention the high voltage power supply is comprised of a piezo-electric crystal and mechanical trigger. Because of the electrode assembly's pipetting feature in the present invention, electrodes can advantageously be spaced more closely than in prior electroporation equipment, thus lower voltages can be employed to achieve the desired field strength. This invention provides a system more compact and safe, yet much less complicated and expensive than other systems, for example U.S. Pat. No. 4,750,100 mentioned above.